Bop1 is a newly characterized nucleolar protein essential for the processing of 28S and 5.8S rRNAs and 60S ribosome biosynthesis (58
). Expression of a dominant negative mutant of Bop1, Bop1Δ, in asynchronous, logarithmically growing cells blocks synthesis of the 60S ribosome subunits and brings about a strong G1
arrest. In the present study, we have characterized the nature of the cell cycle arrest conferred by Bop1Δ and shown that this arrest is dependent on functional p53. These observations implicate the p53 pathway in a heretofore unknown function as a monitor of ribosome biogenesis and provide important insight into the molecular mechanisms linking ribosome biogenesis and cell proliferation in mammalian cells.
Induction of the Bop1 dominant negative mutant in LAP3 cells elicits changes in cell cycle regulators consistent with a G1
checkpoint response. Progression through G1
requires the activity of several cyclin-Cdk complexes (53
). Our results indicate that expression of Bop1Δ causes inhibition of both Cdk4 and Cdk2 activities: (i) their immunoprecipitated complexes display low kinase activity in vitro, and (ii) the lack of hyperphosporylation of pRb suggests that inhibition of the corresponding holoenzyme activities takes place in the cell as well. The Rb family proteins are important targets of cyclin D-Cdk4 and cyclin E/A-Cdk2 (5
). Hyperphosphorylation of pRb is associated with, and partially controls, passage through the restriction point in late G1
, which marks commitment to DNA synthesis (68
). The absence of hyperphosphorylated forms of pRb in Bop1Δ-arrested cells indicates that this critical event does not occur.
The idea that cell cycle progression may depend on some aspect of ribosome biogenesis was first proposed in early studies on the cell cycle (6
), although the nature of this connection has remained unknown. Growth-inhibitory stimuli have been shown to repress RNA polymerase I transcription, suggesting that alterations in ribosome production may thus modulate the potential for cellular proliferation (8
). We infer that ribosome depletion per se is unlikely the only determinant of cell cycle inhibition connected with ribosome biogenesis. When cells are devoid of functional p53, they can progress through G1
to the S phase in spite of the absence of 60S subunit synthesis caused by expression of Bop1Δ (Fig. and ). Conversely, cells bearing functional p53 become arrested in response to Bop1Δ when they possess enough ribosomes for virtually unabated protein synthesis (Fig. ). Thus, defects in ribosome synthesis and inhibition of the cell cycle can be dissociated—when p53 function is impaired, Bop1Δ expression leads to rRNA processing defects but does not induce strong cell cycle arrest (Fig. and ).
Although we cannot rule out the possibility that Bop1Δ directly activates the p53-mediated cell cycle arrest pathway, we favor the idea that expression of Bop1Δ triggers an arrest response indirectly by causing perturbations in rRNA and/or ribosome biogenesis. This model is supported by the observation that DNA synthesis inhibition is delayed compared to inhibition of 28S rRNA maturation (Fig. ), suggesting that the primary effect of Bop1Δ is on rRNA biosynthesis and cell cycle arrest is likely a secondary effect. In addition, previous studies with mammalian cells indicate that interference with ribosome biogenesis by other means can also inhibit cell proliferation. For example, the ts
422E temperature-sensitive mutant of the Syrian hamster cell line BHK21 that is unable to produce mature 28S rRNA and 60S ribosome subunits was rapidly growth arrested at the nonpermissive temperature (38
); the nature of this growth defect, however, has not been clearly defined (20
). Antisense-mediated inhibition of the nucleolar protein p120, a human homolog of the yeast Nop2p that is implicated in biosynthesis of the large ribosome subunit, arrested human lymphocytes in G1
). Recently, conditional deletion of the ribosomal S6 gene was shown to inhibit cell proliferation in the livers of mice (65
). Deletion of S6 using the Cre/LoxP system in this study abrogated production of 40S subunits in liver cells and inhibited their entry into S phase, leading to the suggestion that control mechanisms may have evolved in the cells to recognize lesions in ribosome biogenesis. The similarity of antiproliferative effects observed in our study and these distinctly different experimental systems strongly argues that various defects in ribosome biogenesis may trigger a cell cycle-inhibitory response.
We propose a hypothesis that the signal transmitted to the cell cycle machinery is generated by a mechanism that monitors some aspects of ribosome production in the nucleolus. The most immediate effect of Bop1Δ expression is disruption of several steps in rRNA processing, which prevents formation of mature 60S ribosome subunits (58
). In recent years, it has become apparent that many complex processes in the cell are monitored by checkpoint systems that generate interruptions of the cell cycle when various types of stress are detected (16
). It is conceivable that cells possess a similar checkpoint mechanism to respond to nucleolar stress—perturbations in the nucleolar biosynthetic machinery that produces ribosomes (Fig. ). This idea appears especially appealing because ribosome biogenesis is a complex process that is highly sensitive to various disturbances in cellular metabolism, including various chemical inhibitors (1
), reduced protein synthesis (21
), and starvation (35
). Hence, detection of anomalies in ribosome biogenesis could potentially provide integration of a variety of inputs indicating unfavorable or toxic environmental conditions. In this line of reasoning, the inhibitory effect of nucleolar stress on the cell cycle may represent a built-in protective mechanism to prevent DNA replication under suboptimal metabolic conditions.
FIG. 9 Nucleolar stress model of cell cycle arrest due to perturbation in ribosome biogenesis. Expression of mutant proteins such as Bop1Δ, exposure to chemical inhibitors of synthesis, and maturation of rRNA and other ribosome components in mammalian (more ...)
The nucleolar stress model is consistent with our finding that p53 plays a role in mediating Bop1Δ-induced cell cycle arrest. The tumor suppressor p53 participates in responses to numerous extra- and intracellular stresses (31
). Intriguingly, several components of the p53 pathway are localized, at least transiently, to the nucleolus, including p53 itself (49
), Mdm2 (60
), and p19Arf (32
). Mdm2 and p53 were also detected in complexes containing ribosomal protein L5 and 5S and 5.8S rRNAs (18
). The significance of the connection of p53 with the nucleolus is unclear but has been suggested to involve sequestration from the nucleus, nuclear export, and colocalization with sites of RNA synthesis. Herein we show, for the first time, a functional link between p53 activity and a protein directly involved in rRNA processing and ribosome biogenesis. The association of components of the p53 pathway with the nucleolus may thus reflect a previously unrecognized role of this pathway in monitoring nucleolar function.
One interesting feature of the nucleolar stress model is that it provides a possible mechanism for the antiproliferative effects of a diverse group of metabolic inhibitors, including many clinically important anticancer drugs, that strongly inhibit the rRNA synthesis and processing machinery (1
). At present, the contribution of impaired RNA synthesis and processing to their action is poorly understood. Nevertheless, there is extensive evidence that the antiproliferative properties of 5-fluorouridine and 5-fluorouracil, commonly used in cancer chemotherapy, depend strongly on their effects on RNA metabolism, which can be experimentally separated from other metabolic effects (19
). A large group of ribonucleotide biosynthesis inhibitors were also shown to induce p53-dependent G1
arrest, leading to the suggestion that this effect might be mediated by inhibition of synthesis of some specific RNA molecules (33
). Thus, it would be of particular interest to determine whether molecular mechanisms linked to rRNA processing and ribosome production might mediate cell cycle effects of different chemotherapeutic agents that target RNA.